Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
- Autores
- Renna, Felipe Javier; Herrera Lopez, Malena; Manifava, Maria; Ktistakis, Nicholas; Vaccaro, Maria Ines
- Año de publicación
- 2023
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.
Fil: Renna, Felipe Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina
Fil: Herrera Lopez, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina
Fil: Manifava, Maria. The Babraham Institute; Reino Unido
Fil: Ktistakis, Nicholas. The Babraham Institute; Reino Unido
Fil: Vaccaro, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina - Materia
-
AUTOPHAGY
LC3
INMUNOFLUORESCENCE
PANCREAS - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/227937
Ver los metadatos del registro completo
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Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 ImmunofluorescenceRenna, Felipe JavierHerrera Lopez, MalenaManifava, MariaKtistakis, NicholasVaccaro, Maria InesAUTOPHAGYLC3INMUNOFLUORESCENCEPANCREAShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.Fil: Renna, Felipe Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Herrera Lopez, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Manifava, Maria. The Babraham Institute; Reino UnidoFil: Ktistakis, Nicholas. The Babraham Institute; Reino UnidoFil: Vaccaro, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaJournal of Visualized Experiments2023-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/227937Renna, Felipe Javier; Herrera Lopez, Malena; Manifava, Maria; Ktistakis, Nicholas; Vaccaro, Maria Ines; Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence; Journal of Visualized Experiments; Journal of Visualized Experiments; 194; 4-2023; 1-131940-087XCONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.jove.com/video/65005info:eu-repo/semantics/altIdentifier/doi/10.3791/65005info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:39:12Zoai:ri.conicet.gov.ar:11336/227937instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:39:13.019CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence |
title |
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence |
spellingShingle |
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence Renna, Felipe Javier AUTOPHAGY LC3 INMUNOFLUORESCENCE PANCREAS |
title_short |
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence |
title_full |
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence |
title_fullStr |
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence |
title_full_unstemmed |
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence |
title_sort |
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence |
dc.creator.none.fl_str_mv |
Renna, Felipe Javier Herrera Lopez, Malena Manifava, Maria Ktistakis, Nicholas Vaccaro, Maria Ines |
author |
Renna, Felipe Javier |
author_facet |
Renna, Felipe Javier Herrera Lopez, Malena Manifava, Maria Ktistakis, Nicholas Vaccaro, Maria Ines |
author_role |
author |
author2 |
Herrera Lopez, Malena Manifava, Maria Ktistakis, Nicholas Vaccaro, Maria Ines |
author2_role |
author author author author |
dc.subject.none.fl_str_mv |
AUTOPHAGY LC3 INMUNOFLUORESCENCE PANCREAS |
topic |
AUTOPHAGY LC3 INMUNOFLUORESCENCE PANCREAS |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
dc.description.none.fl_txt_mv |
Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins. Fil: Renna, Felipe Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Herrera Lopez, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Manifava, Maria. The Babraham Institute; Reino Unido Fil: Ktistakis, Nicholas. The Babraham Institute; Reino Unido Fil: Vaccaro, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina |
description |
Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-04 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://purl.org/coar/resource_type/c_6501 info:ar-repo/semantics/articulo |
format |
article |
status_str |
publishedVersion |
dc.identifier.none.fl_str_mv |
http://hdl.handle.net/11336/227937 Renna, Felipe Javier; Herrera Lopez, Malena; Manifava, Maria; Ktistakis, Nicholas; Vaccaro, Maria Ines; Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence; Journal of Visualized Experiments; Journal of Visualized Experiments; 194; 4-2023; 1-13 1940-087X CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/227937 |
identifier_str_mv |
Renna, Felipe Javier; Herrera Lopez, Malena; Manifava, Maria; Ktistakis, Nicholas; Vaccaro, Maria Ines; Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence; Journal of Visualized Experiments; Journal of Visualized Experiments; 194; 4-2023; 1-13 1940-087X CONICET Digital CONICET |
dc.language.none.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
info:eu-repo/semantics/altIdentifier/url/https://www.jove.com/video/65005 info:eu-repo/semantics/altIdentifier/doi/10.3791/65005 |
dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-nd/2.5/ar/ |
eu_rights_str_mv |
openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/ |
dc.format.none.fl_str_mv |
application/pdf application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Journal of Visualized Experiments |
publisher.none.fl_str_mv |
Journal of Visualized Experiments |
dc.source.none.fl_str_mv |
reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
reponame_str |
CONICET Digital (CONICET) |
collection |
CONICET Digital (CONICET) |
instname_str |
Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.name.fl_str_mv |
CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.mail.fl_str_mv |
dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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1844613239988551680 |
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13.070432 |